α-Halomethyl derivatives of amino acids

ABSTRACT

Novel halomethyl derivatives of amino acids of the following general structure ##STR1## wherein Y is FCH 2  --, F 2  CH 2  --, F 3  C--, ClCH 2  -- or Cl 2  CH--; R 1  is hydroxy, a straight or branched alkoxy group of from 1 to 8 carbon atoms, --NR 11  R 12  wherein each of R 11  and R 12  is hydrogen or a straight or branched lower alkyl group of from 1 to 4 carbon atoms, ##STR2## wherein R 5  is hydrogen, a straight or branched lower alkyl group of from 1 to 4 carbon atoms, benzyl or p-hydroxybenzyl; R 2  is hydrogen, alkylcarbonyl wherein the alkyl moiety has from 1 to 4 carbon atoms and is straight or branched, alkoxycarbonyl wherein the alkoxy moiety has from 1 to 4 carbon atoms and is straight or branched or ##STR3## wherein R 6  is hydrogen, a straight or branched lower alkyl group of from 1 to 4 carbon atoms, benzyl or p-hydroxybenzyl; each of R 3  and R 4  is hydrogen, halogen selected from fluorine, chlorine, bromine and iodine, or a straight or branched lower alkyl of from 1 to 4 carbon atoms and may be the same or different with the proviso that when both R 3  and R 4  are halogen R 3  and R 4  are the same; and pharmaceutically acceptable salts thereof.

This is a division, of application Ser. No. 819,975, filed July 28, 1977now U.S. Pat. No. 4,315,095.

FIELD OF INVENTION

This invention relates to novel pharmaceutically useful halomethylderivatives of histidine and related compounds.

BACKGROUND OF INVENTION

Most mammalian tissue contains histamine, concentration beingparticularly high in the skin, intestinal mucosa and the lungs. Everymammalian tissue that contains histamine, including white blood cells,appears capable of synthesizing the amine from histidine. The principalenzyme involved in catalyzing in vivo the conversion of histidine tohistamine is histidine decarboxylase which is specific for the substrateL-histidine. In many tissues the chief storage site of histamine is themast cell, or in the case of blood, the basophil which is thecirculating counterpart of the fixed-tissue mast cell. Mast cells arenot the only tissue source of histamine which is present in substantialamounts in the human epidermis, the central nervous system and thegastrointestinal mucosa.

Histamine is involved in various physiological processes. Histamine isreleased during the antigen-antibody reaction and is responsible, inlarge part, for the hypersensitivity reaction characterized byvasodilation, itching and edema formation. This type of antigen-antibodyreaction wherein the principal cells involved are mast cells andbasophils from which histamine is released is commonly referred to as animmediate hypersensitivity reaction. In addition to antigens, orallergens, histamine is released by many chemical substances,macromolecules, venoms, physical insult, such as heat and otherinjurious stimuli. Gastric acid secretion is known to be stimulated byhistamine. Also, histamine is known to be frequently involved ininitiation of sensory impulses evoking pain and itching. It has alsobeen found that histamine levels are high in many tissues undergoingrapid growth, for example, embryonic tissue, regenerating liver andmalignant growths.

Correlations between levels of histamine and histidine decarboxylaseactivity in tissues have been made. In the brain which containshistamine and histidine decarboxylase the turnover of histamine is rapidbeing augmented by stressful stimuli that also increases histidinedecarboxylase activity. Inhibitors of L-histidine decarboxylase, suchas, α-hydrazinohistidine are known to lower histamine concentrations. Inrat fetal tissue, wherein high levels of histamine are present, it hasbeen shown that inhibition of L-histidine decarboxylase arrests fetaldevelopment.

The effects of histamine and its mode of action are well documented. Itis believed that the amine exerts its effect through at least tworeceptors being classified as H₁ and H₂ receptors. Several agents areknown to counter the effects of histamine, however, not all such agentsprevent the formation of histamine. For example, classicalantihistamines useful in treating allergic reactions are believed toexert their utility by interfering with the binding of histamine with H₁receptors. Similarly agents useful in countering the stimulant effect ofhistamine on gastric acid secretion are believed to operate byinterfering with the binding of histamine with H₂ receptors.

Agents capable of blocking H₁ receptors find use in treating acuteexudative types of allergy, such as, seasonal rhinitis, hay fever,pollinosis relieving the sneezing, rhinorrhea, itching eyes, nose andthroat. Such agents are also useful in controlling cough and to a degreefind use in treating systemic anaphylaxis and bronchial asthma.Antihistamine agents which act through H₁ receptors are also useful intreating allergic dermatoses, such as acute and chronic urticaria,angioedema, itching pruritides, for example, atopic dermatitis andcontact dermatitis, in the control of urticarial and edematous lesionsof serum sickness, control of blood transfusion reactions and control ofdrug reactions attributable to allergic phenomena. Agents which block H₂receptors are useful in treating peptic ulceration, theZollinger-Ellison syndrome and other gastric hypersecretory states.

Agents which block the formation of histamine by inhibiting the activityof histidine decarboxylase, for example, α-methylhistidine andα-hydrazinohistidine, are reported to be useful in the same manner asantihistaminic agents that are blockers of H₁ and H₂ receptors.Additionally histidine decarboxylase inhibitors are implicated as beinguseful in the control of certain tumors which are high in histaminecontent.

The compounds of the present invention prevent the formation ofhistamine by inhibiting the action of histidine decarboxylase renderingsaid compounds useful in treating pathophysiological conditions whichresult from histamine. The presently claimed compounds can be used inthe same manner and for the same purposes as are compounds thatantagonize H₁ and H₂ receptors.

SUMMARY OF INVENTION

The compounds of the present invention are represented by the followinggeneral Formula I: ##STR4## In the above general Formula I Y is FCH₂ --,F₂ CH--, F₃ C--, ClCH₂ -- or Cl₂ CH--; each of R₃ and R₄ is hydrogen,halogen selected from fluorine, chlorine, bromine and iodine or astraight or branched lower alkyl group of from 1 to 4 carbon atoms andcan be the same or different with the proviso that when both R₃ and R₄are halogen R₃ and R₄ are the same; R₁ is hydroxy, a straight orbranched lower alkoxy group of from 1 to 8 carbon atoms, --NR₁₁ R₁₂wherein each of R₁₁ and R₁₂ is hydrogen or a straight or branched loweralkyl group of from 1 to 4 carbon atoms, or ##STR5## wherein R₅ ishydrogen, a straight or branched lower alkyl group of from 1 to 4 carbonatoms, benzyl or p-hydroxybenzyl; R₂ is hydrogen, alkylcarbonyl whereinthe alkyl moiety has from 1 to 4 carbon atoms and is straight orbranched, alkoxycarbonyl wherein the alkoxy moiety has from 1 to 4carbon atoms and is straight or branched or ##STR6## wherein R₆ ishydrogen, a straight or branched lower alkyl group of from 1 to 4 carbonatoms, benzyl or p-hydroxybenzyl.

The pharmaceutically acceptable salts and individual optical isomers ofthe compounds of general Formula I are also included within the scope ofthis invention.

DETAILED DESCRIPTION OF INVENTION

Illustrative examples of straight or branched alkoxy groups of from 1 to8 carbon atoms in the above general Formula I are methoxy, ethoxy,n-propoxy, n-butoxy, isopropoxy, tert-butoxy, pentoxy, and octyloxy.

Illustrative examples of straight chain or branched chain lower alkylgroups of from 1 to 4 carbon atoms in the above general Formula I aremethyl, ethyl, n-propyl, isopropyl, n-butyl and tert-butyl.

As used in general Formula I the term alkylcarbonyl is taken to mean thegroup ##STR7## wherein the alkyl group is straight or branched and hasfrom 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl and tert-butyl.

As used in general Formula I the term alkoxycarbonyl is taken to meanthe group ##STR8## wherein the alkoxy group, that is, --O-alkyl has from1 to 4 carbon atoms and is straight or branched, for example, methoxy,ethoxy, n-propoxy, isopropoxy, n-butoxy and tert-butoxy.

Illustrative examples of pharmaceutically acceptable salts of thecompounds of this invention include non-toxic acid addition salts formedwith inorganic acids, such as, hydrochloric, hydrobromic, sulfuric andphosphoric acid, and organic acids, such as, methane sulfonic,salicylic, maleic, cyclamic, malonic, tartaric, citric and ascorbicacids; and non-toxic salts formed with inorganic or organic bases suchas those of alkali metals, for example, sodium, potassium and lithium,alkaline earth metals, for example, calcium and magnesium, light metalsof Group III A, for example, aluminum, organic amines, such as, primary,secondary or tertiary amines, for example, cyclohexylamine, ethylamine,pyridine, methylaminoethanol, ethanolamine and piperazine. The salts areprepared by conventional means.

Compounds of general Formula I wherein R₂ is hydrogen or alkylcarbonylas defined above, R₁ is hydroxy or a straight or branched alkoxy groupof from 1 to 8 carbon atoms are preferred. More preferred compounds ofthis invention are those of general Formula I wherein R₁ is hydroxy or astraight or branched alkoxy group of from 1 to 8 carbon atoms, R₂ ishydrogen and each of R₃ and R₄ is hydrogen, chlorine, fluorine ormethyl. Compounds wherein Y is FCH₂ --, F₂ CH-- or ClCH₂ -- are alsopreferred.

Illustrative examples of compounds of the present invention are thefollowing:

2-amino-2-difluoromethyl-3-(5-imidazolyl)propionic acid,

2-amino-2-fluoromethyl-3-[5-(4-fluoro)imidazolyl]propionic acid,

2-amino-2-chloromethyl-3-[5-(2-fluoro)imidazolyl]propionic acid,

2-amino-2-dichloromethyl-3-[5-(4-methyl)imidazolyl]propionic acid,

2-amino-2-trifluoromethyl-3-[5-(2-methyl)imidazolyl]propionic acid,

2-amino-2-fluoromethyl-3-[5-(4-ethyl)imidazolyl]propionic acid,

2-amino-2-difluoromethyl-3-[5-(2-ethyl)imidazolyl]propionic acid,

2-amino-2-dichloromethyl-3-[5-(4-n-propyl)imidazolyl]propionic acid,

2-amino-2-chloromethyl-3-[5-(2-n-propyl)imidazolyl]propionic acid,

2-amino-2-dichloromethyl-3-[5-(4-isopropyl)imidazolyl]propionic acid,

2-amino-2-trifluoromethyl-3-[5-(2-isopropyl)imidazolyl]propionic acid,

2-amino-2-dichloromethyl-3-[5-(4-n-butyl)imidazolyl]propionic acid,

2-amino-2-fluoromethyl-3-[5-(2-n-butyl)imidazolyl]propionic acid,

2-amino-2-difluoromethyl-3-[5-(4-tert-butyl)imidazolyl]propionic acid,

2-amino-2-dichloromethyl-3-[5-(2-tert-butyl)imidazolyl]propionic acid,

2-amino-2-fluoromethyl-3-[5-(2,4-difluoro)imidazolyl]propionic acid,

2-amino-2-trifluoromethyl-3-[5-(2,4-dimethyl)imidazolyl]propionic acid,

2-amino-2-difluoromethyl-3-[5-(2,4-diethyl)imidazolyl]propionic acid,

2-amino-2-difluoromethyl-3-(5-(2,4-di-n-propyl)imidazolyl]propionicacid,

2-amino-2-fluoromethyl-3-[5-(2,4-diisopropyl)imidazolyl]propionic acid,

2-amino-2-fluoromethyl-3-[5-(2,4-di-n-butyl)imidazolyl]propionic acid,

2-amino-2-chloromethyl-3-[5-(2,4-di-tert-butyl)imidazolyl]propionicacid,

methyl 2-amino-2-dichloromethyl-3-(5-imidazolyl)propionate, ethyl2-amino-2-trifluoromethyl-3-[5-(4-fluoro)imidazolyl]propionate,

n-propyl 2-amino-2-trifluoromethyl-3-[5-(2-fluoro)imidazolyl]propionate,

N-n-butyl2-amino-2-dichloromethyl-3-[5-(4-methyl)imidazolyl]propionamide,

isopropyl 2-amino-2-chloromethyl-3-[5-(2-methyl)imidazolyl]propionate,

tert-butyl 2-amino-2-fluoromethyl-3-[5-(4-ethyl)imidazolyl]propionate,

n-pentyl 2-amino-2-fluoromethyl-3-[5-(2-ethyl)imidazolyl]propionate,

isopentyl 2-amino-2-chloromethyl-3-[5-(4-n-propyl)imidazolyl]propionate,

tert-pentyl2-amino-2-dichloromethyl-3-[5-(2-n-propyl)imidazolyl]propionate,

n-hexyl2-amino-2-difluoromethyl-3-[5-(4-isopropyl)imidazolyl]propionate,

n-heptyl2-amino-2-difluoromethyl-3-[5-(2-isopropyl)imidazolyl]propionate,

n-octyl 2-amino-2-fluoromethyl-3-[5-(4-n-butyl)imidazolyl]propionate,

methyl 2-amino-2-fluoromethyl-3-[5-(2-n-butyl)imidazolyl]propionate,

N,N'-dimethyl2-amino-2-chloromethyl-3-[5-(4-tert-butyl)imidazolyl]propionamide,

N-n-propyl2-amino-2-trifluoromethyl-3-[5-(2,4-difluoro)imidazolyl]propionamide,

N-n-butyl2-amino-2-difluoromethyl-3-[5-(2,4-dimethyl)imidazolyl]propionamide,

ethyl 2-amino-2-fluoromethyl-3-[5-(2,4-diethyl)imidazolyl]propionate,

n-butyl2-amino-2-fluoromethyl-3-[5-(2,4-di-n-propyl)imidazolyl]propionate,

N-ethyl 2-amino-2-difluoromethyl-3-[5-imidazolyl]propionamide,

N-methyl2-amino-2-trifluoromethyl-3-[5-(4-n-butyl)imidazolyl]propionamide,

N-n-propyl2-amino-2-chloromethyl-3-[5-(2-fluoro)imidazolyl]propionamide,

2-[2-amino-2-dichloromethyl-3-(5-imidazolyl)-1-oxopropylamino]aceticacid,

2-[2-amino-2-fluoromethyl-3-(5-imidazolyl)-1-oxopropylamino]propionicacid,

2-[2-amino-2-difluoromethyl-3-[5-(2-methyl)imidazolyl]-1-oxopropylamino]-2-benzylaceticacid,

2-[2-difluoromethyl-2-(1-oxoethylamino)-3-(5-(4-fluoro)imidazolyl]-1-oxopropylamino)aceticacid,

2-[2-(N-ethoxycarbonylamino)-2-fluoromethyl-3-[5-(4-methyl)imidazolyl]-1-oxopropylamino]aceticacid,

N,N'-diethyl2-dichloromethyl-2-(1-oxoethylamino)-3-[5-(2-fluoro)imidazolyl]propionamide,

2-chloromethyl-2-(1-oxoethylamino)-3-(5-imidazolyl)propionic acid,

2-dichloromethyl-2-(N-propoxycarbonylamino)-3-[5-(2-methyl)imidazolyl]propionicacid,

ethyl2-[N-(2-amino-1-oxoethyl)amino]-2-difluoromethyl-3-(5-imidazolyl)propionate,

N-methyl2-[N-(2-amino-1-oxo-3-phenylpropyl)amino]-2-chloromethyl-3-[5-(2-fluoro)imidazolyl]propionamide,

2-amino-2-chloromethyl-3-[5-(4-chloro)imidazolyl]propionic acid,

2-amino-3-[5-(2-chloro)imidazolyl]-2-difluoromethylpropionic acid,

2-amino-3-[5-(2-chloro-4-methyl)imidazolyl]-2-difluoromethylpropionicacid,

2-amino-3-[5-(2-bromo)imidazolyl]-2-trifluoromethylpropionic acid, and

2-amino-2-fluoromethyl-3-[5-(2-iodo)imidazolyl]propionic acid,

The compounds of this invention, that is, the compounds of Formula I areirreversible inhibitors of histidine decarboxylase, the enzyme which invivo converts histidine to histamine. Thus, the compounds block theformation of histamine which is known to play an important role incertain patho-physiological conditions. As inhibitors of histidinedecarboxylase the compounds of Formula I are useful in the same manneras any known antihistaminic agent whether such agent exerts itseffectiveness by blocking H₁ or H₂ receptors or other means. Thecompounds of this invention are useful in treating patho-physiologicalconditions due to histamine, hence, the compounds have many utilitiesbeing useful in treating acute exudative types of allergy, such as,seasonal rhinitis, hay fever, and pollinosis relieving the sneezing,rhinorrhea, itching eyes, nose and throat. The compounds of generalFormula I are also useful in controlling cough and in treating systemicanaphylaxis and bronchial asthma, and are useful as bronchodilators.Also, the compounds of this invention are useful in treating allergicdermatoses, such as, acute urticaria, chronic urticaria, angioedema,itching pruritides, for example, atopic dermatitis and contactdermatitis. The compounds of this invention are also useful in treatingurticarial and edematous lesions of serum sickness, blood transfusionreactions attributable to allergic phenomena and nausea. The compoundsof general Formula I are also useful in treating peptic ulceration, theZollinger-Ellison syndrome and other gastric hypersecretory states. Asdescribed hereinabove it has been found that histamine levels are highin rapidly growing tissues, such as, tumors, hence, the compounds ofgeneral Formula I by inhibiting the formation of histamine, may beuseful in controlling the growth of certain tumors, for example, Walkermammary carcinoma and Erlich ascitic tumors.

The compounds of Formula I can be administered in various manners toachieve the desired effect. The compounds can be administered alone orin the form of pharmaceutical preparations to the patient being treatedeither orally, parenterally, that is, subcutaneously, intravenously orintraperitoneally, or topically. The compounds can be administered byintranasal instillation or by application to mucous membranes such asthat of the nose, throat and bronchial tubes, for example, in an aerosolspray containing small particles of a novel compounds of this inventionin a spray solution or dry powder form.

The amount of novel compound of this invention administered will varyand can be any effective amount. Depending on the patient, the conditionbeing treated and the mode of administration, the quantity of novelcompound administered may vary over a wide range to provide as aneffective amount in a unit dosage form of from about 0.1 to 500 mg/kg(milligrams per kilogram) of body weight of the patient per dose andpreferably from about 50 to 200 mg/kg to achieve the desired effect. Forexample, the desired effect can be obtained by consumption of a unitdosage form, such as, for example, a tablet containing from 10 to 500 mgof a novel compound of this invention taken 1 to 4 times daily.

As used herein the term patient is taken to mean warm blooded animalssuch as birds and mammals, for example, cats, dogs, rats, mice, guineapigs, sheep, horses, bovine cows, and humans.

The solid unit dosage forms can be of the conventional type. Thus, thesolid form can be a capsule which can be of the ordinary gelatin typecontaining a novel compound of this invention and a carrier, forexample, lubricant and inert fillers such as lactose, sucrose and cornstarch. In another embodiment, the novel compounds are tableted withconventional tablet bases such as lactose, sucrose or corn starch incombination with binders such as acacia, corn starch or gelatin,disintegrating agents, such as corn starch, potato starch, or alginicacid, and a lubricant such as stearic acid, or magnesium stearate.

For parenteral administration the compounds of Formula I may beadministered as injectable dosages of a solution or suspension of thecompound in a physiologically acceptable diluent with a pharmaceuticalcarrier which can be a sterile liquid such as water and oils with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. Illustrative of oils which can be employed inthese preparations are those of petroleum, animal, vegetable orsynthetic origin, for example, peanut oil, soybean oil, and mineral oil.In general water, saline, aqueous dextrose, and related sugar solutions,ethanols and glycols such as propylene glycol or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

The compounds of Formula I can be administered in the form of a depotinjection or implant preparation which may be formulated in such amanner as to permit a sustained release of the active ingredient. Theactive ingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers or synthetic silicones, for example, Silastic, silicone rubbermanufactured by the Dow-Corning Corporation.

For use as aerosols the novel compounds of Formula I in solution orsuspension may be packaged in a pressurized aerosol container togetherwith a gaseous or liquefied propellant, for example,dichlorodifluoromethane, dichlorodifluoromethane withdichlorodifluoroethane, carbon dioxide, nitrogen or propane, with theusual adjuvants such as cosolvents, and wetting agents, as may benecessary or desirable. The compounds may also be administered in anon-pressurized form such as in a nebulizer or atomizer.

The utility of the compounds of general Formula I as irreversibleinhibitors of histidine decarboxylase may be demonstrated as follows. Acompound of general Formula I is administered as an aqueous solution orsuspension to rats or mice either orally or parenterally. At differenttime intervals after administration of the test compound the animals areinjected intraperitoneally with 2 μCi of 2-¹⁴ C-L-histidine. Two hoursafter the labeled histidine injection the animals are sacrificed, andthe amount of radio active histamine present in the glandular part ofthe stomach is determined as described by K. M. Mole and D. M. Shepherd,J. Pharm. Pharmac. 25, 609-613 (1973).

In addition to being useful pharmacological agents, the compounds ofFormula I wherein R₁ is hydroxy and R is hydrogen are useful asintermediates for the preparation of cephalosporin derivatives of thefollowing general Formula II which are useful as antibiotics. ##STR9##In the above general Formula II X is hydrogen or acetoxy; M is hydrogenor a negative charge, and R₃, R₄ and Y have the meanings defined ingeneral Formula I.

The compounds of general Formula II and the pharmaceutically acceptablesalts and individual optical isomers thereof are novel compounds usefulas antibiotics and can be administered in a manner similar to that ofmany well known cephalosporin derivatives, for example, cephalexin,cephalothin, or cephaloglycine. The compounds of general Formula II andpharmaceutically acceptable salts and isomers thereof can beadministered alone or in the form of pharmaceutical preparations eitherorally or parenterally and topically to warm blooded animals, that is,birds and mammals, for example, cats, dogs, bovine cows, sheep, horsesand humans. For oral administration the compounds can be administered inthe form of tablets, capsules or pills or in the form of elixirs orsuspensions. For parenteral administration, the compounds may best beused in the form of a sterile aqueous solution which may contain othersolutes, for example, enough saline or glucose to make the solutionisotonic. For topical administration the compounds of general FormulaII, salts and isomers thereof may be incorporated into creams orointments.

Illustrative examples of bacteria against which the compounds of generalFormula II and the pharmaceutically acceptable salts and individualoptical isomers thereof are active are Staphylococcus aureus, Salmonellaschotmuehleri, Klebsiella pneumoniae, Diplococcus pneumoniae andStreptococcus pyogenes.

Illustrative pharmaceutically acceptable non-toxic inorganic acidaddition salts of the compounds of general Formula II are mineral acidaddition salts, for example, hydrogen chloride, hydrogen bromide,sulfates, sulfamates, phosphate, and organic acid addition salts are,for example, maleate, acetate, citrate, oxalate, succinate, benzoate,tartrate, fumarate, malate and ascorbate. The salts can be formed byconventional means.

Illustrative examples of cephalosporin derivatives as represented bygeneral Formula II are7-[[2-amino-2-difluoromethyl-3-(5-imidazolyl)propionyl]amino]-3-acetyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid,7-[[2-amino-2-fluoromethyl-3-[2-(methyl)-5-imidazolyl]propionyl]amino]-3-acetyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid and7-[[2-amino-2-difluoromethyl-3-[2-fluoro-5-imidazolyl]propionyl]amino]3-acetyloxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid.

The preparation of the compounds of general Formula II is describedhereinbelow.

The α-halomethyl histidine derivatives of this invention wherein R₁ ishydroxy, R₂ is hydrogen and R₃ and R₄ have the meanings defined ingeneral Formula I are prepared by treating one equivalent of a suitablyprotected histidine derivative of the formula ##STR10## with oneequivalent of a suitable strong base to generate a carbanionintermediate followed by treatment with one equivalent of a suitablehalomethylhalo alkylating reagent and subsequently hydrolyzing withacid. In the above general Formula III R_(a) is a straight or branchedalkyl group having from 1 to 8 carbon atoms, R_(b) is hydrogen, phenyl,a straight or branched alkyl group of from 1 to 8 carbon atoms, methoxyor ethoxy; R_(c) is phenyl or a straight or branched alkyl group of from1 to 8 carbon atoms; or R_(b) and R_(c) taken together form an alkylenegroup of from 5 to 7 carbon atoms, that is, --CH₂ --(CH₂)_(m) --CH₂ --wherein m is an integer of from 3 to 5. Illustrative examples ofstraight or branched alkyl groups of from 1 to 8 carbon atoms whichR_(b) and R_(c) may represent are methyl, ethyl, n-propyl, isopropyl,n-butyl, tert-butyl, n-hexyl, n-octyl, and neopentyl. Each of R₃ and R₄has the meaning defined in general Formula I.

Suitable strong bases which may be employed in the above reactionsequence to form the carbanion intermediate are those which willabstract a proton from the carbon atom alpha to the carboxy group suchas, alkyl lithium, for example, butyl lithium or phenyl lithium, lithiumdialkylamide, for example, lithium diisopropylamide, or lithium amide,tertiary potassium butylate, sodium amide, metal hydrides, for example,sodium hydride or potassium hydride, tertiary amines, such as,triethylamine, lithium acetylide or dilithium acetylide. Lithiumacetylide, dilithium acetylide, sodium hydride and lithiumdiisopropylamide are preferred bases.

Suitable halomethylhalo alkylating reagents which may be employed in theabove reaction are illustratively chlorofluoromethane,bromofluoromethane, fluoroiodomethane, chlorodifluoromethane,bromodifluoromethane, difluorodomethane, bromotrifluoromethane,chlorotrifluoromethane, trifluoroidomethane, bromochloromethane,dichloromethane, chloroiodomethane, bromidichloromethane anddichloroiodomethane. The halomethylhalo alkylating reagents are known inthe art.

The alkylating reaction may be carried out in an aprotic solvent, forexample, benzene, toluene, ethers, tetrahydrofuran, dimethylsulfoxide orhexamethylphosphortriamide. The reaction temperature may vary from about-120° C. to about 65° C., a preferred reaction temperature being about25° to 55° C. The reaction time will vary from about 1/2 hour to 24hours.

Acid hydrolysis to remove any unreacted starting material and protectinggroups may be achieved in one step or stepwise. In a one-step hydrolysisprocedure the concentration of acid employed will obviously vary withthe duration of the hydrolysis step and temperature employed. Forexample, one-step hydrolysis may be achieved by treatment withconcentrated hydrochloric acid for 1 to 4 days at about 25° to 120° C.Stepwise hydrolysis may be achieved by treatment with dilute acid forabout 1/2 hour to 6 hours at about 25° C. to remove unreacted startingmaterial repeating treatment with dilute acid to remove any amineprotecting groups followed by treatment with concentrated acid for about1 to 3 days at about 25° to 125° C. to remove any ester or ether groups.Stepwise hydrolysis is preferred.

The compounds of general Formula III are prepared by treating anappropriately substituted N'-monotritylhistidine lower alkyl ester, suchas, the methyl, ethyl or n-propyl ester in a solvent, such as, an ether,for example, diethyl ether or tetrahydrofuran, aromatic hydrocarbons,such as, benzene, methylene chloride or chloroform with a carbonylbearing compound to form a Schiff's base in a generally known manner,specifically (a) when R_(b) is hydrogen, by treating the appropriateamino acid ester with benzaldehyde or an alkanol having from 1 to 9carbon atoms being straight or branched, for example, 1-propanal,1-butanal, 2,2-dimethylpropan-1-al or 2,2-diethylbutan-1-al, (b) whenR_(b) is phenyl by treating the appropriate amino acid ester withbenzophenone or phenyl alkyl ketone wherein the alkyl moiety has from 1to 8 carbon atoms and is straight or branched, for example, phenylmethyl ketone, phenyl ethyl ketone, phenyl isopropyl ketone, phenyln-butyl ketone or phenyl tert-butyl ketone, and (c) when R_(b) is astraight or branched alkyl group having from 1 to 8 carbon atoms,treating the appropriate amino acid ester with a phenyl alkyl ketone asdescribed above with a dialkyl ketone wherein each alkyl moiety has from1 to 8 carbon atoms and is straight or branched, for example, dimethylisopropyl ketone, di-n-butyl ketone or methyl tert-butyl ketone. Thecarbonyl bearing compounds are known in the art or may be prepared byprocedures well known in the art.

When R_(b) is methoxy or ethoxy in compounds of Formula III anappropriate amino acid ester is reacted with benozyl halide, forexample, chloride or an alkanoic acid halide, for example, chloridewherein the alkanoic acid has from 1 to 9 carbon atoms and may bestraight or branched, such as acetyl chloride, propionyl chloride,butyryl chloride, tert-butyryl chloride, 2,2-diethylbutyric acidchloride or valeryl chloride at 0° C. in ethers, methylenechloride,dimethylformamide, dimethylacetamide, or chlorobenzene in the presenceof an organic base such as triethylamine or pyridine after which thereaction mixture is allowed to warm to about 25° C. for 1 hour. Theresulting amide derivative is combined with an alkylating reagent, suchas, methylfluorosulfonate, dimethylsulfate, methyliodide, methyl,p-toluenesulfonate or trimethyloxonium hexafluorophosphate when R_(b) lis methoxy or triethyloxonium tetrafluoroborate when R_(b) is ethoxy atabout 25° C. in a chlorinated hydrocarbon solvent, such as, methylenechloride, chlorobenzene or chloroform, and the reaction mixture isrefluxed for about 12 to 20 hours. The mixture is then cooled to about25° C. and an organic base, such as, triethylamine or pyridine is addedafter which the solution is extracted with brine and the productisolated.

When in the compounds of Formula III R_(b) and R_(c) together form analkylene group of from 5 to 7 carbon atoms said amino acid esterderivatives are obtained by treating the amino acid ester with a cyclicalkanone selected from cyclopentanone, cyclohexanone and cycloheptanoneto form a Schiff's base by procedures generally known in the art.

The appropriately substituted N'-monotritylhistidine lower alkyl estersare prepared from the corresponding ester hydrochlorides by treatmentwith a saturated solution of sodium bicarbonate, and the free base isextracted with chloroform. The tritylester hydrochlorides are preparedfrom histidine or histidine substituted at the 2 and/or 4-positions witha halogen selected from fluorine, chlorine, bromine or iodine or a loweralkyl group of from 1 to 4 carbon atoms and the 2- and 4-positionsubstituents can be the same or different with the proviso that whenboth the 2- and 4-position substituents are halogen they are the same bythe general methods described by G. C. Stelakatos et al., J. Am. Chem.Soc. 81, 2884 (1959). The 2,4-substituted histidine derivatives areknown in the art or can be prepared by procedures generally known in theart, for example, as illustrated in the specific examples containedherein.

The compounds of general Formula I wherein R₁ is a straight or branchedalkoxy group of from 1 to 8 carbon atoms are prepared from thecorresponding derivatives wherein R₁ is hydroxy by reaction with analcohol of the formula R₇ --OH wherein R₇ is a straight or branchedalkyl group of from 1 to 8 carbon atoms, such as, methyl, ethyl,n-propyl, isopropyl, n-butyl, tert-butyl, hexyl or octyl, saturated withHCl gas at about 25° C. for from 12 to 36 hours.

The compounds of general Formula I wherein R₁ is --NR₁₁ R₁₂ wherein eachof R₁₁ and R₁₂ is hydrogen or a lower alkyl group of from 1 to 4 carbonatoms are prepared by an acylation reaction of an acid halide, forexample, an acid chloride, of the corresponding compound wherein each R₁is hydroxy and R₂ has the meaning defined in Formula I with the provisothat any free amino group is suitably protected with, for example,carbobenzyloxy or tert-butoxycarbonyl, and the 2-imidazole nitrogen isprotected with carbobenzyloxy or 2,4-dinitrophenyl, with an excess of anappropriate amine which may be represented as HNR₁₁ R₁₂. The reaction iscarried out in methylene chloride, chloroform, dimethylformamide ethers,such as, tetrahydrofuran or dioxane, or benzene at about 25° C. forabout 1 to 4 hours. Suitable amines are ammonia, or a compound which isa potential source of ammonia, for example, hexamethylenetetramine;primary amines, for example, methylamine, ethylamine or n-propylamine;and secondary amines, for example, dimethylamine, diethylamine ordi-n-butylamine. Following the acylation reaction the protecting groupsare removed by treatment with acid, for example, trifluoroacetic acid orhydrogen bromide in dioxane followed by treatment with base such asaqueous sodium hydroxide.

The compounds of general Formula I wherein R₁ is ##STR11## are preparedby reacting the corresponding derivative wherein R₁ is hydroxy or afunctional derivative thereof such as an acid anhydride and R₂ has themeaning defined in Formula I with the proviso that any free amino groupis protected with a suitable blocking group, such as benzyloxycarbonylor tert-butoxycarbonyl and the 1-imidazole nitrogen is protected withbenzyloxycarbonyl or 2,4-dinitrophenyl with a compound of the formula##STR12## wherein R₅ has the meaning defined in general Formula I and R₈is a lower alkyl group, for example, methyl or ethyl in an ether, forexample, tetrahydrofuran or dioxane at 0° to 50° C. for about 1 to 24hours followed by acid and base hydrolysis to remove the protectinggroups, with the proviso that when the amine protected free acid isemployed the reaction is carried out using a dehydrating agent such asdicyclohexylcarbodiimide.

The compounds of general Formula I wherein R₂ is alkylcarbonyl whereinthe alkyl moiety is straight or branched and has from 1 to 4 carbonatoms are prepared by treating the corresponding derivative wherein R₁is hydroxy and R₂ is hydrogen with an acid halide of the formula##STR13## wherein halo is a halogen atom, for example, chlorine orbromine and R₉ is a straight or branched alkyl group having from 1 to 4carbon atoms in water in the presence of a base such as sodium hydroxideor sodium borate at a temperature of from about 0° to 25° C. for fromabout 1/2 hour to 6 hours. These compounds may also be prepared from theester derivative, that is, compounds of general Formula I wherein R₂ ishydrogen and R₁ is an alkoxy group of from 1 to 8 carbon atoms bytreatment with the acid halide, ##STR14## described above, in water,methylene chloride, chloroform or dimethyl acetamide in the presence ofa base such as sodium hydroxide, potassium hydroxide or excesstriethylamine at a temperature of from 0° to 25° C. for from 1/2 hour to24 hours.

The compounds of general Formula I wherein R₂ is alkoxycarbonyl whereinthe alkoxy moiety is straight or branched and has from 1 to 4 carbonatoms are prepared by treating the corresponding derivative wherein R₂is hydrogen and R₁ is hydroxy with a halo alkylformate of the formula##STR15## wherein halo is halogen atom such as chlorine or bromine andR₁₀ is a straight or branched alkyl group having from 1 to 4 carbonatoms in water in the presence of a base such as sodium hydroxide orsodium borate at a temperature of from about 0° to 25° C. for from about1/2 hour to 6 hours.

The compounds of general Formula I wherein R₂ is ##STR16## wherein R₆ ishydrogen, a straight or branched lower alkyl group of from 1 to 4 carbonatoms, benzyl or p-hydroxybenzyl are prepared by treating thecorresponding derivative wherein R₂ is hydrogen, R₁ is a straight orbranched alkoxy group of from 1 to 8 carbon atoms and the 1-imidazolenitrogen is protected with benzyloxycarbonyl or 2,4-dinitrophenyl withan acid of the formula ##STR17## or an anhydride thereof wherein theamino group is protected with a suitable blocking group such asbenzyloxycarbonyl or tert-butoxycarbonyl and R₆ has the meaning definedhereinabove in an ether, such as, tetrahydrofuran or dioxane, methylenechloride or chloroform, and in the presence of a dehydrating agent suchas dicyclohexylcarbodiimide when the free acid is employed, at atemperature of from about 0° C. to 35° C. for about 1 to 12 hoursfollowed by acid and base hydrolysis to remove the protecting groups.

The individual optical isomers of the compounds of Formula I wherein R₂is H and R₁ is OH may be separated by using a (+) or (-)binaphthylphosphoric acid salt by the method of R. Viterbo et al.,Tetrahedron Letters 48, 4617 (1971). The individual optical isomers ofcompounds wherein R₂ and R₁ are other than H and OH respectively areprepared as described herein for the racemic mixture only starting withthe resolved amino acid.

As set forth hereinabove the compounds of general Formula I wherein R₁is hydroxy and R₂ is hydrogen are useful as intermediates for thepreparation of useful cephalosporin derivatives as described by generalFormula II. The compounds of general Formula II are prepared by coupling7-aminocephalosporanic acid or a derivative thereof having the formula##STR18## wherein X and M have the meaning defined in general Formula IIwith an acid of the following Formula V or a functional derivativethereof, such as, the acid chloride or an acid anhydride ##STR19##wherein Y, R₃ and R₄ have the meanings defined in Formula I and theamino groups are protected by suitable blocking groups, for example, anacid salt, such as hydrochloride salt, an acyl group, ortert-butoxycarbonyl which groups are removed after the coupling reactionby acid hydrolysis.

The coupling reaction is generally carried out in a solvent, such as,ethyl acetate, dioxane, chloroform or tetrahydrofuran in the presence ofa base, such as alkaline bicarbonate. The temperature of the reactionmay vary from -10° C. to 100° C., and the reaction time may very fromabout 1/2 hour to 10 hours. The cephalosporin products are isolated byconventional means.

The following illustrates the use of the compounds of general Formula Iwherein R₁ is hydroxy in the preparation of the useful cephalosporinderivatives of general Formula II.

EXAMPLE 17-[[2-Amino-2-difluoromethyl-3-(5-imidazolyl)propionyl]amino]-3-acetoxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid

A mixture of 1 g of3-acetyloxymethyl-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid and 1 g of 2-amino-2-difluoromethyl-3-(5-imidazolyl)propionic acidchloride wherein the free amino groups are protected withtertiary-butoxycarbonyl in 50 ml of ethyl acetate is refluxed for 2hours after which the solvent is removed leaving a residue which ischromatographed on silica gel using benzene-acetone as the eluant togive7-[[2-amino-2-difluoromethyl-3-(5-imidazolyl)propionyl]amino]-3-acetoxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid wherein the amino groups are protected with tert-butoxycarbonyl.The protected cephalosporin compound is treated with trifluoroaceticacid for 1/2 hour at 25° C. under nitrogen atmosphere then diluted withether until precipitation stops and filtered to give thedi-trifluoroacetic acid salt of the title cephalosporin which can beconverted to free base by use of ion exchange resin.

The following examples are illustrative of pharmaceutical preparationsof compounds of general Formula I.

EXAMPLE 2

An illustrative composition for hard gelatin capsules is as follows:

    ______________________________________                                        (a)   2-amino-2-difluoromethyl-3-(5-imid-                                                                   100    mg                                             azolyl)propionic acid                                                   (b)   talc                    20     mg                                       (c)   lactose                 500    mg                                       ______________________________________                                    

The formulation is prepared by passing the dry powders of (a) and (b)through a fine mesh screen and mixing them well. The powder is thenfilled into hard gelatin capsules at a net fill of 620 mg per capsule.

EXAMPLE 3

An illustrative composition for tablets is as follows:

    ______________________________________                                        (a)   2-amino-2-difluoromethyl-3-(5-imid-                                                                   200    mg                                             azolyl)propionic acid                                                   (b)   starch                  50     mg                                       (c)   lactose                 100    mg                                       (d)   magnesium stearate      5      mg                                       ______________________________________                                    

The granulation obtained upon mixing the lactose with the compound (a)and the part of the starch and granulated with starch paste is dried,screened, and mixed with the magnesium stearate. The mixture iscompressed into tablets weighing 355 mg each.

EXAMPLE 4

An illustrative composition for an aerosol solution is the following:

    ______________________________________                                                            Weight percent                                            ______________________________________                                        (a)   2-amino-2-difluoromethyl-3-[5-(2-                                                                 20.0                                                      fluoro)imidazolyl]propionic acid                                        (b)   ethanol             30.0                                                (c)   dichlorodifluoromethane                                                                           50.0                                                ______________________________________                                    

The materials (a), (b) and (c) are packaged in 15 ml stainless steelcontainers equipped with a metering valve designed to meter 0.2 gramsper dose, an equivalent of 40 mg of novel compound (a).

EXAMPLE 5

An illustrative composition for an injedtable suspension is thefollowing 1 ml ampul for an intramuscular injection.

    ______________________________________                                                            Weight percent                                            ______________________________________                                        (a)   2-amino-2-fluoromethyl-3-[5-(4-                                                                   5.0                                                       methyl)imidazolyl]propionic acid                                        (b)   polyvinylpyrrolidone                                                                              0.5                                                       (M.W. 25000)                                                            (c)   lecithin            0.25                                                (d)   water for injection to make                                                                       100.0                                               ______________________________________                                    

The materials (a)-(d) are mixed, homogenized, and filled into 1 mlampuls which are sealed and autoclaved 20 minutes at 121° C. Each ampulcontains 50 mg per ml of novel compound (a).

EXAMPLE 6

An illustrative composition for an aerosol suspension is the following:

    ______________________________________                                                          Weight percent                                              ______________________________________                                        (a)   2-amino-2-chloromethyl-3-[5-                                                                    20.0                                                        imidazolyl]propionic acid                                                     (particly size <10μ)                                                 (b)   sorbitan trioleate                                                                              0.5                                                   (c)   dichlorodifluoromethane                                                                         39.75                                                 (d)   dichlorodifluoroethane                                                                          39.75                                                 ______________________________________                                    

The materials (a)-(d) are packaged in 15 ml stainless steel containersequipped with a metering value designed to meter 50 mg per dose, anequivalent of 10 mg of novel compound (a).

EXAMPLE 7 Methyl2-benzaldimine-2-difluoromethyl-3-[5-(1-trityl)imidazolyl]propionate

Methyl 2-amino-3-(1-trityl-5-imidazolyl)propionate hydrochloride (1.52g, 34 mmoles) is treated with a saturated solution of sodiumbicarbonate, and the free base is extracted with chloroform. To asolution of the oily 2-amino-3-(1-trityl-5-imidazolyl)propionate (1.31g, 32 mmole) in 20 ml of methylene chloride is added 337 mg (0.032 M) ofbenzaldehyde with stirring. Stirring is continued for about 15 hours atabout 25° C. after which the mixture is concentrated to an oily residuewhich is crystallized in ether to give the Schiff's base, methyl2-benzaldimine-3-(1-trityl-5-imidazolyl)propionate, m.p. 132° C. To asolution of lithium diisopropylamide (3.5 mmole), prepared fromdiisopropylamine and n-butyllithium, in 20 ml of tetrahydrofuran cooledto -78° C. magnetically stirred and kept under nitrogen, is added slowly1.5 g (3 mmoles) of the above-obtained Schiff's base in 18 ml oftetrahydrofuran. The reaction mixture is allowed to warm to roomtemperature and is then rapidly warmed to 55° C. The nitrogen inlet isreplaced by a balloon filled with chlorodifluoromethane (about 300 ml).After addition of the chlorodifluoromethane the reaction mixture is keptat room temperature for about 12 hours then quenched with brine. Etherextraction gives methyl2-benzaldimine-2-difluoromethyl-3-[5-(1-trityl)imidazolyl]propionate asa yellow and light powder.

EXAMPLE 8 2-Amino-2-difluoromethyl-3-(5-imidazolyl)propionic acidhydrochloride

A suspension of methyl2-benzaldimine-2-difluoromethyl-3-[5-(1-trityl)imidazolyl]propionate(1.5 g, 2.7 mmole) in concentrated hydrochloric acid (15 ml) is heatedat 90° C. for 15 hours. The reaction mixture is evaporated to dryness,diluted with water and carefully extracted with chloroform. Theresulting oil is dissolved in dry ethanol (3 ml) and a solution oftriethylamine N in ethanol is added until the pH reaches 3.5. Themixture is evaporated to dryness and washed several times withchloroform. The resulting oil is decolorized with charcoal and twocrystallizations from water-ethanol yields2-amino-2-difluoromethyl-3-(5-imidazolyl)propionic acid hydrochloride,m.p. 250° C.

EXAMPLE 9 Methyl 2-difluoromethyl-2-amino-3-(5-imidazolyl)propionate

To a solution of crude methyl2-difluoromethyl-2-benzaldimine-3-(1-trityl-5-imidazolyl)propionate (13g) in ether (200 ml) is added a solution of HCl 0.5 M (10 ml). Themixture is stirred at room temperature for 12 hours after which theorganic phase is separated and concentrated. The residue (12 g) is takenup in chloroform (150 ml) and treated with concentrated HCl (50 ml) atroom temperature for 12 hours. The aqueous phase is separated andconcentrated under reduced pressure to give methyl2-amino-2-difluoromethyl-3-(5-imidazolyl)propionate dihydrochloride.

EXAMPLE 10 2-Amino-2-difluoromethyl-3-(5-imidazolyl)propionic acidhydrochloride

A solution of methyl 2-amino-2-difluoromethyl-3-(5-imidazolyl)propionatedihydrochloride (10 g) in concentrated HCl is heated at refluxtemperature for 16 hours. Concentration of the reaction mixture gives aresidue which is passed on an Amberlite IR 120 H⁺ form resin column.Elution with a solution of HCl 2 M afforded the2-amino-2-difluoromethyl-3-(5-imidazolyl)propionic acid hydrochloridewhich is recrystallized from water/ethanol.

When in the procedure of Example 7 an appropriate amount of thehydrochloride of methyl2-amino-3-[5-(2-fluoro-1-trityl)imidazolyl]propionate, methyl2-amino-3-[5-(4-fluoro-1-trityl)imidazolyl]propionate, methyl2-amino-3-[5-(2-methyl-1-trityl)imidazolyl)propionate, methyl2-amino-3-[5-(4-methyl-1-trityl)imidazolyl]propionate, methyl2-amino-3-[5-(2,4-difluoro-1-trityl)imidazolyl]propionate, methyl2-amino-3-[5-(2-fluoro-4-methyl-1-trityl)imidazolyl]propionate or methyl2-amino-3-[5-(2,4-dichloro-1-trityl)imidazolyl]propionate is substitutedfor methyl 2-amino-3-[5-(1-trityl)imidazolyl]propionate the followingrespective compounds are obtained:

methyl2-benzaldimine-2-difluoromethyl-3-[5-(2-fluoro-1-trityl)imidazolyl]propionate,

methyl2-benzaldimine-2-difluoromethyl-3-[5-(4-fluoro-1-trityl)imidazolyl]propionate,

methyl2-benzaldimine-2-difluoromethyl-3-[5-(2-methyl-1-trityl)imidazolyl]propionate,

methyl2-benzaldimine-2-difluoromethyl-3-[5-(4-methyl-1-trityl)imidazolyl]propionate,

methyl2-benzaldimine-2-difluoromethyl-3-[5-(2,4-difluoro-1-trityl)imidazolyl]propionate,and

methyl2-benzaldimine-3-[5-(2-fluoro-4-methyl-1-trityl)imidazolyl]propionate.

When an appropriate amount of the above-obtained imidazolyl propionatederivatives is substituted for methyl2-benzaldimine-2-difluoromethyl-3-[5-(1-trityl)imidazolyl]propionate inthe procedure of Example 8 the following respective products areobtained:

2-amino-2-difluoromethyl-3-[5-(2-fluoro)imidazolyl]propionate acid,

2-amino-2-difluoromethyl-3-[5-(4-fluoro)imidazolyl]propionic acid,

2-amino-2-difluoromethyl-3-[5-(2-methyl)imidazolyl]propionic acid,

2-amino-2-difluoromethyl-3-[5-(4-methyl)imidazolyl]propionic acid,

2-amino-2-difluoromethyl-3-[5-(2,4-difluoro)imidazolyl]propionic acid,and

2-amino-2-difluoromethyl-3-[5-(2-fluoro-4-methyl)imidazolyl]propionicacid.

When in the procedure of Example 7 an appropriate amount ofdichloromethane or chlorofluoromethane is substituted forchlorodifluoromethane methyl2-benzaldimine-2-chloromethyl-3-[5-(1-trityl)imidazolyl]propionate andmethyl2-benzaldimine-2-fluoromethyl-3-[5-(1-trityl)imidazolyl]propionate areobtained which when substituted in appropriate amounts for methyl2-benzaldimine-2-difluoromethyl-3-[5-(1-trityl)imidazolyl]propionate inthe in the procedure of Example 8 gives respectively2-amino-2-chloromethyl-3-(5-imidazolyl)propionic acid, m.p. 116° C.(dec.) and 2-amino-2-fluoromethyl-3-(5-imidazolyl)propionic acid.

EXAMPLE 11 N-Acetyl 2-amino-2-fluoromethyl-3-(5-imidazolyl)propionicacid

A solution of 1.87 g (0.01 mole) of2-amino-2-fluoromethyl-3-(5-imidazolyl)propionic acid in 10 ml of 2 Nsodium hydroxide solution is cooled to 5° C. To this solution,maintained at 5° C., are added simultaneously from two syringes 1.0 g(0.013 mole) of acetyl chloride and 26 ml 2 N sodium hydroxide dropwise.After 2 hours the solution is neutralized by the addition of 8 ml of 5 Nhydrochloric acid followed by 0.5 ml of acetic acid. The mixture iscooled to 0° C. and the resultant N-acetyl2-amino-2-fluoromethyl-3-(5-imidazolyl)propionic acid filtered off.

EXAMPLE 12 Methyl 2-amino-2-fluoromethyl-3-(5-imidazolyl)propionatedihydrochloride

A suspension of 4.4 g (0.024 mole) of2-amino-2-fluoromethyl-3-(5-imidazolyl)propionic acid in 30 ml ofmethanol at 0° C. is saturated with dry hydrogen chloride after whichthe solution is heated under reflux for 12 hours. The solution is thenconcentrated and allowed to crystallize to afford methyl2-amino-2-fluoromethyl-3-(5-imidazolyl)propionate, dihydrochloride (4.0g).

EXAMPLE 13 Ethyl N-acetyl2-amino-2-fluoromethyl-3-(5-imidazolyl)propionate monohydrochloride

A solution of 2.29 g (0.01 mole) of N-acetyl2-amino-2-fluoromethyl-3-(5-imidazolyl)propionic acid in 50 ml ofethanol is saturated with dry hydrogen chloride, then heated underreflux overnight. The solution is concentrated then cooled and the ethylester monohydrochloride filtered off.

EXAMPLE 142(2-Aminopropionamido)-2-fluoromethyl-3-(5-imidazolyl)propionic acid

A solution of 2.72 g (0.01 mole) of methyl2-amino-2-fluoromethyl-3-(5-imidazolyl)propionate dihydrochloride and2.1 g (0.02 mole) of triethylamine in 40 ml of dichloromethylene at 25°C. is treated with 3.4 g (0.02 mole) of benzyl chloroformate and 2.1 gof triethylamine. After 2 hours at 25° C. the solution is washed withwater, dried and concentrated to afford the dicarbobenzoxy methyl ester.To this residue is added 30 ml of 40% (w/w) hydrogen bromide in dioxaneand the mixture allowed to stand at 25° C. for 30 minutes. Ether (150ml) is then added and the resultant precipitate is filtered off andadded to cold bicarbonate solution then rapidly extracted intodichloromethane. The dichloromethane solution is dried and concentratedto afford methylN-carbobenzoxy-2-amino-2-fluoromethyl-3-(5-imidazolyl)propionate (2.3 g,0.007 mole) which is treated in 10 ml of dichloromethane with 1.6 g(0.007 mole) of N-carbobenzoxy alanine and 1.45 g (0.007 mole) ofN,N'-dicyclohexylcarbodimide overnight at 25° C. The mixture is thencooled to 0° C., the precipitated dicyclohexyl urea filtered off, theorganic solution washed with 1 N hydrochloric acid, bicarbonatesolution, then dried and concentrated. The residue is then treated with30 ml of 40% (w/w) hydrogen bromide in dioxane for 30 minutes at 25° C.Addition of 150 ml of ether resulted in a precipitate of thehydrobromide which was filtered off and treated overnight with 50 ml of1 N sodium hydroxide at 25° C. The resulting solution is adjusted toneutral pH and the product isolated from an Amberlite 120 H⁺ resin byelution with ammonia (1 M).

EXAMPLE 15 N-(2-Propionicacid)-2-amino-2-fluoromethyl-3-(5-imidazolyl)propionyl carboxamide

To a solution of 1.87 g (0.01 mole) of2-amino-2-fluoromethyl-3-(5-imidazolyl)propionic acid in 5 ml of 4 Nsodium hydroxide is added 10 ml of 5% sodium carbonate and 8 ml ofdioxane. The solution is cooled in an ice bath and treated with 3.9 g(0.021 mole) of benzyl chloroformate and 5 ml of 4 N sodium hydroxide,added dropwise and simultaneously from two dropping funnels. The mixtureis stirred for an additional 20 minutes at 0° C., then 30 ml of ethylacetate is added and the reaction mixture is neutralized with 6 Nhydrochloric acid. The aqueous phase is extracted with ethyl acetate,the organic solutions combined, dried and concentrated to about 5 ml.Ether (15 ml) is added and theN,N'-dicarbobenzoxy-2-amino-2-fluoromethyl-3-(5-imidazolyl)propionicacid (2.9 g, 0.007 mole) filtered off. The N,N'-dicarbobenzoxyderivative is dissolved in 15 ml of methylene chloride and treated with720 mg (0.007 mole) of alanine methyl ester in 5 ml of methylenechloride and 1.45 g (0.007 mole) of N,N'-dicyclohexylcarbodimide. Themixture is allowed to stand overnight at 25° C., then refrigerated andthe precipitated dicyclohexyl urea filtered off. The filtrate is washedwith N hydrochloric acid followed by 5% bicarbonate, then dried andconcentrated to afford N-(2-propionic acid methylester)-N,N'-dicarbobenzoxy-3-amino-2-fluoromethyl-3-(5-imidazolyl)propionylcarboxamide which is dissolved in dioxane containing 15 ml of 40% (w/w)hydrogen bromide and allowed to stand at room temperature for 30minutes. Ether (100 ml) is added and the resulting precipitate collectedand suspended in 50 ml of 0.8 N NaOH and stirred overnight at 25° C. Theaqueous solution is extracted with ether, neutralized, and applied to anAmberlite 120 H⁺ resin and the product isolated by elution with ammonia(1 M).

EXAMPLE 16 N-(n-Propyl)-2-amino-2-fluoromethyl-3-(5-imidazolyl)propionylcarboxamide, dihydrochloride

N,N'-Dicarbobenzoxy-2-amino-2-fluoromethyl-3-(5-imidazolyl)propionicacid (2.9 g, 0.007 mole), prepared as described for N-(2-propionicacid)-2-amino-2-fluoromethyl-3-(5-imidazolyl)propionyl carboxamide, in30 ml of dichloromethane is treated with 82.5 mg (0.007 mole) of thionylchloride at 25° C. for one hour. Propylamine (820 mg, 0.014 mole) isadded and the solution stirred at 25° C. for another hour. The solutionis then washed with water, dried and evaporated. The residue is treatedwith 15 ml of a solution of dioxane containing hydrogen bromide (40%w/w) and ethanol and allowed to stand for 30 minutes at 25° C. Ether (50ml) is then added, the resulting precipitate collected and treated with200 ml of 1 N sodium hydroxide and 20 ml of methanol overnight at 25° C.The solvents are concentrated by evaporation and the solution extractedwith dichloromethane. The organic phase is in turn extracted with 1 NHCl and the aqueous phase evaporated to afford the product.

EXAMPLE 17 2-Fluoro-4-methylhistidine

4-Methylhistidine methyl ester (free base) (10 g) is added to 35 ml oftrifluoroacetic anhydride at -10° C. The mixture is stirred at 25° C.for 5 hours, then the solvent is removed under reduced pressure. Water(50 ml) is added then removed in vacuo, this procedure being repeated 3times, followed by a similar treatment with ethanol. The residue isrecrystallized from ethyl acetate-ether to affordα-N-trifluoroacetyl-4-methylhistidine methyl ester as thetrifluoroacetate salt.

A solution of 1.44 g (200 mM) of NaNO₂ in 20 ml of water at 0° C. isadded to a solution of 2.44 g (0.02 mole) of p-bromoaniline in 300 ml of2.3 N HCl at 0° C. After 40 minutes at 0° C. the resulting solution ofthe diazonium salt is added to a solution ofα-N-trifluoroacetyl-4-methyl histidine methyl ester (5.5 g, 0.1 M, fromthe trifluoroacetate salt) in 200 ml of 0.2 M aqueous sodium carbonate.After 2 hours at 0° C. the orange precipitate (6.0 g) is collected anddried, then suspended in 200 ml of ethanol containing 0.5 g of platinumoxide and subjected to catalytic hydrogen (25° C., 40 psi Paar bomb)overnight after which the catalyst is filtered off and the solventevaporated. The residue is treated with 100 ml of water, filtered andwashed with ether. The aqueous layer is evaporated and the residuerecrystallized from ethanol-ether to affordα-N-trifluoroacetyl-2-amino-4-methylhistidine methyl ester.

To 100 ml of 50% aqueous HBF₄ at -10° C. is addedα-N-trifluoroacetyl-2-amino-4-methylhistidine methyl ester (2.5 g, 8.6mM), followed by NaNO₂ (0.8 g, 12 mM) in 5 ml of water. This solution isdiluted with 100 ml of cold HBF₄ and irradiated at 0° C. with a Hanovia450-W medium-pressure mercury vapor lamp, equipped with a Corex filter,in a quartz immersion well. After 1 hour irradiation time the reactionmixture is neutralized to pH 6 with cold concentrated sodium hydroxide,then extracted with ethylacetate. The ethyl acetate solution isconcentrated to afford an oily residue which is treated with 15 ml of0.5 M sodium hydroxide for 4 hours at 25° C. After neutralization thesolution is applied to an Amberlite 120 H⁺ resin, and the product elutedwith NH₄ OH (1 M). Evaporation of the ammonia and recrystallization frommethanol gives 2-fluoro-4-methylhistidine.

EXAMPLE 18 4-Fluoro-2-methylhistidine

2-Methylhistidine methyl ester (free base) (10 g) is added to 35 ml oftrifluoroacetic anhydride at -10° C. The mixture is stirred at 25° C.for 5 hours, then the solvent is removed under reduced pressure. Water(50 ml) is added and then removed in vacuo, this procedure beingrepeated 3 times, followed by a similar treatment with ethanol. Theresidue is recrystallized from ethyl acetate-ether to affordα-N-trifluoroacetyl-2-methylhistidine methyl ester as itstrifluoroacetate salt.

A solution of 1.44 g (200 mM) of NaNO₂ in 20 ml of water at 0° C. isadded to a solution of 3.44 g (0.02 mole) of p-bromoaniline in 100 ml of2.3 N HCl at 0° C. After 40 minutes at 0° C. the resulting solution ofthe diazonium salt is added to a solution ofα-N-trifluoroacetyl-4-methyl histidine methyl ester (5.5 g, 0.1 M, fromthe trifluoroacetate salt) in 200 ml of 0.2 M aqueous sodium carbonate.After 2 hours at 0° C. the orange precipitate (6.0 g) is collected anddried, then suspended in 200 ml of ethanol containing 0.5 g platinumoxide and subjected to catalytic hydrogen (25° C., 40 psi Paar bomb)overnight. The catalyst is filtered off and the solvent evaporated. Theresidue is treated with 100 ml of water, filtered and washed with ether.The aqueous layer is evaporated and the residue recrystallized fromethanol-ether to afford α-N-trifluoroacetyl-2-methyl-4-aminohistidinemethyl ester.

To 100 ml of 50% aqueous HBF₄ at -10° C. is addedα-N-trifluoroacetyl-2-methyl-4-aminohistidine methyl ester (2.5 g, 8.6mM) followed by NaNO₂ (0.8 g, 12 mM) in 5 ml of water. This solution isdiluted with cold HBF₄ (100 ml) and irradiated at 0° C. with a Hanovia450-W medium-pressure mercury vapor lamp, equipped with a Corex filter,in a quartz immersion well. After 1 hour irradiation time the reactionmixture is neutralized to pH 6 with cold concentrated sodium hydroxide,then extracted with ethyl acetate. The ethyl acetate solution isconcentrated to afford an oily residue which is treated with 15 ml of0.5 M sodium hydroxide for 4 hours at 25° C. After neutralization thesolution is applied to an Amberlite 120 H⁺ resin, and the product elutedwith NH₄ OH (1 M). Evaporation of the ammonia and recrystallization frommethanol gives 4-fluoro-2-methylhistidine.

EXAMPLE 19 2,4-Difluorohistidine

A solution of 1.44 g (200 mM) of NaNO₂ in water (20 ml) at 0° C. isadded to a solution of p-bromoaniline (6.88 g, 0.04 M) in HCl (200 ml of2.3 N) at 0° C. After 40 minutes at 0° C. the resulting solution of thediazonium salt is added to a solution of α-N-benzoylhistidine methylester (5.5 g, 0.1 M) in aqueous Na₂ CO₃ (400 ml of 0.2 M). After 2 hoursat 0° C. the orange precipitate (12.0 g) is collected and dried, thensuspended in 400 ml of ethanol containing 0.5 g of platinum oxide andsubjected to catalytic hydrogen (25° C., 40 psi Paar bomb) overnight.The catalyst is filtered off and the solvent evaporated. The residue istreated with 200 ml of water, filtered and washed with ether. Theaqueous layer is evaporated and the residue recrystallized fromethanol-ether to afford α-N-benzoyl-2,4-diaminohistidine methyl ester.

To 100 ml of 50% aqueous HBF₄ at -10° C. is addedα-N-benzoyl-2,4-diamino histidine methyl ester (2.5 g), followed byNaNO₂ (1.6 g, 24 mM) in water (10 ml). This solution is diluted withcold HBF₄ (200 ml) and irradiated at 0° C. with a Hanovia 450-Wmedium-pressure mercury vapor lamp, equipped with a Corex filter, in aquartz immersion well. After 1 hour irradiation time the reactionmixture is neutralized to pH 6 with cold concentrated sodium hydroxide,then extracted with ethyl acetate. The ethyl acetate solution isconcentrated to afford an oily residue which is treated with NaOH (0.5M, 30 ml) for 14 hours at 25° C. After neutralization the solution isapplied to an Amberlite 120 H⁺ resin, and the product eluted with NH₄ OH(1 M). Evaporation of the ammonia and recrystallization from methanolgives 2,4-difluorohistidine.

The 2-alkyl or 4-alkyl and the 2,4-dialkylhistidine derivatives employedherein are prepared from 5-hydroxymethylimidazole substituted at the 2and/or 4-positions with an appropriate lower alkyl group by the generalprocedure described by K. Matsumoto et al., Agr. Biol. Chem. 38 (5),1097 (1974). The 2,4-dialkyl-5-hydroxymethylimidazoles are prepared byhydroxymethylation of the 2,4-dialkylimidazole with formaldehyde by thegeneral procedure of M. Masui et al., Chem. Pharm. Bull. 1974, 2359. The4-alkyl-5-hydroxymethylimidazoles are similarly prepared according tothe method described by Ewins, J. Chem. Soc. 99, 2052 (1911). The2-alkyl-5-hydroxymethylimidazoles are prepared by hydroxymethylation of1-benzyl-2-alkylimidazoles to give1-benzyl-2-alkyl-4-hydroxymethylimidazoles according to E. F. Godefroiet al., Rec. Trav. Chim. Pays Bas 91, 1385 (1972). The N-benzyl group issubsequently removed using Na/NH₃ as described, for example, by R. G.Jones, J. Am. Chem. Soc. 71, 383 (1949).

EXAMPLE 20 2,4-Dichlorohistidine

To a solution of 1.6 g (24 mM) of sodium nitrite in 16 ml ofconcentrated sulfuric acid at 25° C. is added 2.5 g (8.6 mM) ofα-N-benzoyl-2,4-diaminohistidine in 20 ml of acetic acid at such a rateas to maintain the temperature below 35° C. This solution is then addedwith cooling to a solution of 10 g of cuprous chloride in 20 ml ofconcentrated hydrochloric acid. The combined solution is maintained at25° C. for 10 minutes then neutralized by careful addition of coldconcentrated sodium hydroxide followed by extraction with ethyl acetate.The ethyl acetate extract is concentrated to afford an oily residuewhich is treated with 30 ml of 0.5 M sodium hydroxide for 14 hours at25° C. After neutralization the solution is applied to an Amberlite 120H⁺ resin and eluting with 1 ammonium hydroxide and evaporation of theammonia affords 2,4-dichlorohistidine upon recrystallization frommethanol.

When in the above procedure of Example 20 an appropriate amount ofcuprous bromide is substituted for cuprous chloride and hydrobromic acidis used in place of hydrochloric acid, 2,4-dibromohistidine is obtained.

The 2,4-diiodohistidine compound [D. Mackay and D. M. Shepherd, Brit. J.Pharmacol. 15, 552 (1960)] is obtained from the diazonium salt formed insitu in the procedure of Example 20, by pouring said salt into aqueouspotassium iodide containing aqueous iodide.

When in the above procedure of Example 20 an appropriate amount of theα-N-benzoyl derivative of 2-amino-4-(lower)alkylhistidine methyl ester,2-(lower)alkyl-4-aminohistidine methyl ester, wherein the lower alkylgroup has from 1 to 4 carbon atoms and is straight or branched,2-aminohistidine or 4-aminohistidine is substituted forα-N-benzyl-2,4-diaminohistidine the following compounds are obtained:

2-chloro-4-(lower)alkylhistidine,

2-(lower)alkyl-4-chlorohistidine,

2-chlorohistidine and

4-chlorohistidine,

and when the procedure of Example 20 is further modified by substitutingan appropriate amount of cuprous bromide for cuprous chloride andhydrobromic acid is substituted for hydrochloric acid the followingcompounds are obtained: 2-bromo-4(lower)alkylhistidine,2-(lower)alkyl-4-bromohistidine, 2-bromohistidine and 4-bromohistidine.

The 2- or 4-diodohistidine derivatives and the 2-iodo-2-(lower)alkylhistidine derivatives are obtained from the 2- or 4-diazoniumhistidinederivative and the 2-diazonium-4-(lower)alkylhistidine derivatives bypouring the appropriate diazonium derivative into aqueous potassiumiodide containing aqueous iodide. The 2- or 4-diazoniumhistidinederivative and the 2-diazonium-2-(lower)alkylhistidine derivatives areformed in situ when in the procedure of Example 20 the α-N-benzoylderivative of 2- or 4-aminohistidine, 2-amino-4-(lower)alkylhistidine or4-amino-2-(lower)alkylhistidine is substituted respectively forα-N-benzoyl-2,4-diaminohistidine.

We claim:
 1. A compound of the formula ##STR20## wherein Y is F₂ CH--,F₃ C--, or Cl₂ CH--; each of R₃ and R₄ is hydrogen, halogen selectedfrom fluorine, chlorine, bromine and iodine or a straight or branchedlower alkyl group of from 1 to 4 carbon atoms and can be the same ordifferent with the proviso that when both R₃ and R₄ are halogen R₃ andR₄ are the same; R₁ is hydroxy, a straight or branched alkoxy group offrom 1 to 8 carbon atoms, --NR₁₁ R₁₂ wherein each of R₁₁ and R₁₂ ishydrogen or a straight or branched lower alkyl group of from 1 to 4carbon atoms or ##STR21## wherein R₅ is hydrogen, a straight or branchedlower alkyl group of from 1 to 4 carbon atoms, benzyl orp-hydroxybenzyl; R₂ is hydrogen, alkylcarbonyl wherein the alkyl moietyhas from 1 to 4 carbon atoms and is straight or branched, alkoxycarbonylwherein the alkoxy moiety has from 1 to 4 carbon atoms and is straightor branched or ##STR22## wherein R₆ is hydrogen, a straight or branchedlower alkyl group of from 1 to 4 carbon atoms, benzyl orp-hydroxybenzyl; and pharmaceutically acceptable salts and individualoptical isomers thereof.
 2. A compound of claim 1 wherein R₂ is hydrogenor alkylcarbonyl wherein the alkyl moiety has from 1 to 4 carbon atomsand is straight or branched.
 3. A compound of claim 1 wherein R₁ ishydroxy or a straight or branched alkoxy group of from 1 to 8 carbonatoms.
 4. A compound of claim 1 wherein each of R₃ and R₄ is hydrogen,chlorine, fluorine or methyl.
 5. A compound of claim 4 wherein R₁ ishydroxy or a straight or branched alkoxy group of from 1 to 8 carbonatoms and R₂ is hydrogen.